Molybdenum disulfide loaded quantum dot shell nanosheet, preparation method thereof, and application in immunochromatography

Abstract

The present invention provides a molybdenum disulfide-loaded quantum dot shell nanosheet, its preparation method and application in immunochromatography, belonging to the technical field of nanomaterial detection probes. The present invention uses a single-layer MoS2 two-dimensional nanosheet as a multifunctional two-dimensional platform to provide a large surface area, high stability and a strong colorimetric signal; polyethyleneimine is used as an electropositive linker to anchor carboxylated quantum dots on the molybdenum disulfide nanosheet, and the resulting quantum dot shell can enhance the fluorescence ability of the nanosheet and provide rich surface sites for antibody modification. The molybdenum disulfide-loaded quantum dot shell nanosheet provided by the present invention has fluorescence quenching ability and inner filtering effect. The loaded shell can produce dual-enhanced colorimetric / fluorescence performance through layer-by-layer self-assembly. MoS2 as a substrate has better stability and a larger reaction interface than ordinary spherical nanomaterials, and can be used for immunochromatographic analysis to achieve qualitative and quantitative detection of target viruses.

Description

Technical Field

[0001] The present invention relates to the technical field of nanomaterial detection probes, and in particular to a molybdenum disulfide-loaded quantum dot shell nanosheet, a preparation method thereof, and an application thereof in immunochromatography. Background Art

[0002] Immunochromatographic assay (ICA) is considered the most promising point-of-care (POCT) technology for the detection of infectious pathogens. However, conventional ICA kits still face inherent drawbacks such as low sensitivity, limited quantitative capability, and reliance on colloidal gold (AuNPs) to provide a visual colorimetric signal. Quantum dots (QDs) have been shown to be ideal fluorescent labels for ICA applications due to their excellent optical properties, including quantifiable fluorescence, narrow emission, broad excitation, and good photostability. However, these QD nanocomposites generally lack strong colorimetric capabilities to support naked-eye determination and require additional UV equipment or reader devices to measure their fluorescence signals. This requirement is a burden in areas with poor medical conditions and reduces the popularity of ICA technology.

[0003] Molybdenum disulfide (MoS2) nanosheets are typical two-dimensional (2D) film-like transition metal dichalcogenides with unique physical and chemical properties, including strong catalytic activity, high band gap tunability and quenching ability, ultrathin structure, and good biocompatibility. They show great promise as a platform for building high-performance biosensors. However, due to the high fluorescence quenching ability and inner filtering effect of 2D MoS2 nanosheets, introducing fluorescence enhancement ability into 2D MoS2 nanosheets remains a great challenge. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a molybdenum disulfide-loaded quantum dot shell nanosheet, a preparation method thereof, and an application in immunochromatography. The molybdenum disulfide-loaded quantum dot shell nanosheet provided by the present invention can be used as a signal label to realize colorimetric-fluorescence dual-signal mode immunochromatographic analysis.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a molybdenum disulfide loaded quantum dot shell nanosheet, comprising a MoS2 two-dimensional nanosheet core and several layers of quantum dot shell wrapped on the surface of the core, wherein the single-layer quantum dot shell comprises a polyethyleneimine layer and carboxylated quantum dots distributed on the surface of the polyethyleneimine layer.

[0007] Preferably, the MoS2 two-dimensional nanosheet has a sheet diameter of 400 to 800 nm;

[0008] The thickness of the single polyethyleneimine layer is 1 to 10 nm.

[0009] Preferably, the carboxylated quantum dots are one or more of carboxylated CdSe / ZnS quantum dots, carboxylated InP / ZnS quantum dots, and carboxylated carbon quantum dots;

[0010] The quantum dot shell has 1 to 4 layers.

[0011] The present invention provides a method for preparing the above-mentioned molybdenum disulfide-loaded quantum dot shell nanosheets, comprising the following steps:

[0012] (1) providing a single-layer MoS2 two-dimensional nanosheet dispersion;

[0013] (2) ultrasonically mixing the single-layer MoS2 two-dimensional nanosheet dispersion with a polyethyleneimine aqueous solution to obtain MoS2@PEI nanosheets;

[0014] (3) ultrasonically mixing the MoS2@PEI nanosheets and carboxylated quantum dots to obtain MoS2-QD composite nanosheets;

[0015] (4) Ultrasonic mixing the MoS2-QD composite nanosheets with the polyethyleneimine aqueous solution again, and repeating the process of steps (2) to (3) to obtain molybdenum disulfide-loaded quantum dot shell nanosheets.

[0016] Preferably, in step (2), the concentration of the polyethyleneimine aqueous solution is 0.2 to 5 mg / mL; the mass ratio of the single-layer MoS2 two-dimensional nanosheet to the polyethyleneimine is 1 to 10:1;

[0017] In the step (3), the molar ratio of the mass of the MoS2 two-dimensional nanosheets to the carboxylated quantum dots is 10 mg:1 to 20 nmol.

[0018] The present invention provides the use of the above-mentioned molybdenum disulfide-loaded quantum dot shell nanosheets in non-diagnostic immunochromatographic analysis and detection.

[0019] The present invention provides a monkeypox virus detection probe, comprising the above-mentioned molybdenum disulfide-loaded quantum dot shell nanosheet and an anti-monkeypox virus antibody chemically coupled to the molybdenum disulfide-loaded quantum dot nanosheet.

[0020] The present invention provides a monkeypox virus detection kit, comprising a detection test strip and the above-mentioned monkeypox virus detection probe;

[0021] The test paper comprises a sample pad, an NC membrane and an absorption pad. The NC membrane is provided with a T line and a C line. The surface of the T line is coated with monkeypox virus capture antibodies, and the surface of the C line is coated with goat anti-mouse antibodies.

[0022] The present invention provides a non-diagnostic method for detecting monkeypox virus in a colorimetric-fluorescence dual-signal mode, comprising the following steps:

[0023] Mixing the monkeypox virus detection probe, a buffer solution, and a sample to be tested to obtain a test solution;

[0024] Add the test solution to the sample pad of the test paper, observe the color of the T line on the surface of the NC membrane after standing, compare the color depth of the T line with a predetermined colorimetric card, and obtain the content of monkeypox virus in the test sample; the colorimetric card is a colorimetric image of the T line on the test paper with different concentrations of monkeypox virus protein;

[0025] Alternatively, the test solution is added to the sample pad of the test paper, and the fluorescence intensity of the T line is tested after standing. The content of monkeypox virus in the test sample is obtained according to the fluorescence intensity and a predetermined standard curve; the standard curve is a linear relationship curve between the monkeypox virus content and the fluorescence intensity.

[0026] Preferably, the color of the T line on the surface of the NC film is observed under the condition that the surface of the NC film is irradiated with ultraviolet light.

[0027] The present invention provides a molybdenum disulfide-loaded quantum dot shell nanosheet (abbreviated as MoS2-MQDs), comprising a MoS2 two-dimensional nanosheet core and several layers of quantum dot shell wrapped around the surface of the core. The single-layer quantum dot shell includes a polyethyleneimine layer and carboxylated quantum dots distributed on the surface of the polyethyleneimine layer. In the present invention, the MoS2 two-dimensional nanosheet serves as a multifunctional two-dimensional platform, providing a large surface area, high stability, and a strong colorimetric signal; the cationic polymer polyethyleneimine (PEI) serves as an electropositive linker, capable of anchoring a large number of carboxylated quantum dots to the MoS2 nanosheet; and the several layers of quantum dot shell containing thousands of carboxylated quantum dots can enhance the fluorescence ability of the nanosheet and provide abundant surface sites for antibody modification. The molybdenum disulfide-loaded quantum dot shell nanosheets provided by the present invention have fluorescence quenching ability and inner filtering effect, and have good dispersibility. The loaded shell can produce dual-enhanced colorimetric / fluorescence performance through layer-by-layer self-assembly. The MoS2 as a substrate has better stability and a larger reaction interface than ordinary spherical nanomaterials, and can be used for immunochromatographic analysis (ICA) to achieve qualitative and quantitative determination of target viruses.

[0028] The present invention provides a method for preparing the above-mentioned molybdenum disulfide-loaded quantum dot shell nanosheets. The present invention uses electrostatic adsorption, specifically, by coating positively charged PEI on the surface of a single-layer MoS2 nanosheet through ultrasound to modify its surface electrical properties, and firmly assembles negatively charged carboxylated quantum dots under the action of electrostatic adsorption, to prepare multi-layer core-shell structured molybdenum disulfide-loaded quantum dot shell nanosheets. This method is simple to operate, low in cost, and suitable for industrial mass production.

[0029] The present invention provides the use of the aforementioned molybdenum disulfide-loaded quantum dot shell nanosheets in non-diagnostic immunochromatographic assays. By integrating MoS2-MQDs into a colorimetric-fluorescence dual-signal immunochromatographic assay, the present invention enables stable, sensitive, and specific detection of target analytes in complex real-world samples. This method has great application potential and can replace traditional point-of-care detection methods to meet the detection needs of various situations. It also significantly improves the sensing range and sensitivity of ICA strips, enabling direct and accurate detection in complex real-world samples. It can serve as a universal immunoassay tool for monkeypox virus and other highly pathogenic viruses.

[0030] The present invention provides a colorimetric-fluorescence dual signal mode detection method for monkeypox virus for non-diagnostic purposes. This method can directly output a colorimetric signal similar to colloidal gold immunochromatography, and can also provide a highly sensitive fluorescence signal, and achieve highly sensitive quantitative detection with the assistance of a portable fluorescence reader. The colorimetric mode can support rapid screening of monkeypox virus without the use of any special equipment, making ultra-sensitive, quantitative and real-time monitoring of the target virus possible. The results of the embodiment show that the visual limit of detection (vLOD) of the colorimetric mode for detecting monkeypox virus antigen is 0.1 ng / mL; under ultraviolet light excitation, the visual sensitivity of the fluorescence signal on the T line to the target antigen is 0.01 ng / mL when measured by naked eye; the fluorescence LOD of the fluorescence signal mode for detecting monkeypox virus antigen is 0.0024 ng / mL, and the relative standard deviation (RSD) of the fluorescence intensity on the T line is less than 8.75%, which has high stability and reliability. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The preparation process of MoS2-MQDs composite nanomaterials and the principle of colorimetric-fluorescence dual-signal immunochromatographic detection;

[0032] Figure 2 This is a microscopic picture of MoS2-MQDs composite nanomaterial;

[0033] Figure 3 This is the fluorescence emission spectrum characterization diagram of MoS2-MQDs composite nanomaterials;

[0034] Figure 4 is the fluorescence intensity of MoS2-MQDs composite nanomaterials at different pH values;

[0035] Figure 5 Characterize the performance of MoS2 composite nanotags with different number of layers;

[0036] Figure 6 Colorimetric images, fluorescence images and corresponding fluorescence signals on the T-line of the monkeypox virus protein detection strip at different concentrations;

[0037] Figure 7 To characterize the detection performance of MoS2-MQDs-ICA;

[0038] Figure 8 These are the test results of throat swabs and lake water samples spiked with viral antigens. DETAILED DESCRIPTION

[0039] The present invention provides a molybdenum disulfide loaded quantum dot shell nanosheet, comprising a MoS2 two-dimensional nanosheet core and several layers of quantum dot shell wrapped on the surface of the core, wherein the single-layer quantum dot shell comprises a polyethyleneimine layer and carboxylated quantum dots distributed on the surface of the polyethyleneimine layer.

[0040] In the present invention, the MoS2 two-dimensional nanosheet preferably has a sheet diameter of 400 to 800 nm, more preferably 500 to 600 nm; and a sheet thickness of 1 to 5 nm, more preferably 2 to 4 nm. In the present invention, the MoS2 two-dimensional nanosheet is preferably a single-layer MoS2 two-dimensional nanosheet.

[0041] In the present invention, the average molecular weight of the polyethyleneimine is preferably 10,000 to 80,000, more preferably 30,000 to 50,000. In the present invention, the thickness of a single polyethyleneimine layer is preferably 1 to 10 nm, more preferably 3 to 8 nm.

[0042] In the present invention, the carboxylated quantum dots are preferably water-soluble quantum dots, specifically preferably one or more of carboxylated CdSe / ZnS quantum dots, carboxylated InP / ZnS quantum dots, and carboxylated carbon quantum dots. In the present invention, the particle size of the carboxylated quantum dots is preferably 10 to 20 nm, more preferably 12 to 16 nm. In the present invention, the source of the carboxylated quantum dots is preferably commercially available. In the present invention, the commercial manufacturer of the carboxylated quantum dots is preferably Suzhou Xingshuo Nanotechnology Co., Ltd.

[0043] In the present invention, the number of layers of the multiple quantum dot shells is preferably 1 to 4, more preferably 2 to 3; the number of carboxylated quantum dots in the multiple quantum dot shells is preferably several thousand, specifically preferably 1,000 to 10,000.

[0044] The present invention provides a method for preparing the above-mentioned molybdenum disulfide-loaded quantum dot shell nanosheets, comprising the following steps:

[0045] (1) providing a single-layer MoS2 two-dimensional nanosheet dispersion;

[0046] (2) ultrasonically mixing the single-layer MoS2 two-dimensional nanosheet dispersion with a polyethyleneimine aqueous solution to obtain MoS2@PEI nanosheets;

[0047] (3) ultrasonically mixing the MoS2@PEI nanosheets and carboxylated quantum dots to obtain MoS2-QD composite nanosheets;

[0048] (4) Ultrasonic mixing the MoS2-QD composite nanosheets with the polyethyleneimine aqueous solution again, and repeating the process of steps (2) to (3) to obtain molybdenum disulfide-loaded quantum dot shell nanosheets.

[0049] The present invention first provides a dispersion of single-layer MoS2 two-dimensional nanosheets. In the present invention, the concentration of the single-layer MoS2 two-dimensional nanosheets in the dispersion is preferably 0.1 to 10 mg / mL, more preferably 1 to 5 mg / mL.

[0050] In the present invention, the method for preparing the single-layer MoS2 two-dimensional nanosheet dispersion preferably includes the following steps:

[0051] The single-layer MoS2 two-dimensional nanosheets were ultrasonically mixed with water, the nanosheets with a diameter of less than 300 nm in the supernatant were discarded, and the remaining precipitate was resuspended in water.

[0052] In the present invention, the water is preferably deionized water. In the present invention, the power of the ultrasonic mixing is preferably 500W, and the time is preferably 10 minutes.

[0053] The present invention ultrasonically mixes the monolayer MoS2 two-dimensional nanosheet dispersion with a polyethyleneimine aqueous solution to produce MoS2@PEI nanosheets. In the present invention, the concentration of the polyethyleneimine aqueous solution is preferably 0.2 to 5 mg / mL, more preferably 2 mg / mL; and the mass ratio of the monolayer MoS2 two-dimensional nanosheet to the polyethyleneimine is preferably 1 to 10:1, more preferably 3 to 6:1.

[0054] In the present invention, the power of the ultrasonic mixing is preferably 500 W, and the time is preferably 20 to 60 minutes, more preferably 30 minutes. During the ultrasonic mixing process, the positively charged polyethyleneimine is wrapped around the surface of the MoS2 two-dimensional nanosheets to form a polyethyleneimine shell.

[0055] In the present invention, after the ultrasonic mixing, the ultrasonic mixture is preferably centrifuged and washed to remove excess polyethyleneimine, and the residue is resuspended in water. In the present invention, the number of centrifugal washings is preferably 3 times.

[0056] After obtaining the MoS2@PEI nanosheets, the present invention ultrasonically mixes the MoS2@PEI nanosheets with carboxylated quantum dots to obtain MoS2-QD composite nanosheets. In the present invention, the molar ratio of the mass of the MoS2 two-dimensional nanosheets to the carboxylated quantum dots is preferably 10 mg:1 to 20 nmol, more preferably 10 mg:10 nmol.

[0057] In the present invention, the power of the ultrasonic mixing is preferably 500 W, and the time is preferably 20 to 60 min, more preferably 40 min.

[0058] In the present invention, after the ultrasonic mixing, the ultrasonic mixture is preferably centrifuged and washed to remove excess carboxylated quantum dots, and the residue is resuspended in water. In the present invention, the number of centrifugal washings is preferably 2 times.

[0059] After obtaining the MoS2-QD composite nanosheets, the present invention further ultrasonically mixes the MoS2-QD composite nanosheets with an aqueous solution of polyethyleneimine, and repeats steps (2) to (3) to obtain molybdenum disulfide-loaded quantum dot shell nanosheets. In the present invention, the number of repetitions depends on the number of layers of the quantum dot shells.

[0060] The present invention provides the use of the aforementioned molybdenum disulfide-loaded quantum dot shell nanosheets in non-diagnostic immunochromatographic assays. By integrating MoS2-MQDs into a colorimetric-fluorescence dual-signal immunochromatographic assay, the present invention enables stable, sensitive, and specific detection of target analytes in complex real-world samples. This method has great application potential and can replace traditional point-of-care detection methods to meet the detection needs of various situations. It also significantly improves the sensing range and sensitivity of ICA strips, enabling direct and accurate detection in complex real-world samples. It can serve as a universal immunoassay tool for monkeypox virus and other highly pathogenic viruses.

[0061] The present invention provides a monkeypox virus detection probe comprising the aforementioned molybdenum disulfide-loaded quantum dot-shelled nanosheets and an anti-monkeypox virus antibody chemically coupled to the molybdenum disulfide-loaded quantum dot-shelled nanosheets. In the present invention, the anti-monkeypox virus antibody is a commercially available antibody, specifically a monoclonal anti-MPXV A29 protein antibody, catalog number 40891-M001740891-M0027, purchased from Beijing Sino Biological Technology Co., Ltd.

[0062] In the present invention, the quantum dots in the molybdenum disulfide-loaded quantum dot shell nanosheets are modified with carboxyl groups, which can be chemically coupled with the side chain amino groups of the basic amino acids in the anti-monkeypox virus antibody.

[0063] In the present invention, the method for preparing the monkeypox virus detection probe preferably comprises the following steps:

[0064] activating the carboxyl groups of the MoS2-loaded quantum dot shell nanosheets to obtain MoS2-loaded quantum dot shell nanosheets with activated carboxyl groups;

[0065] The activated carboxyl molybdenum disulfide-loaded quantum dot shell nanosheets are mixed with anti-monkeypox virus antibodies and MES buffer solution, and incubated to obtain a monkeypox virus detection probe.

[0066] In the present invention, the carboxyl group activating agent used for the carboxyl group activation is preferably EDC and NHS.

[0067] In the present invention, the pH value of the MES buffer solution is 6 and the concentration is 10 mM.

[0068] In the present invention, the incubation temperature is preferably room temperature, and the incubation time is preferably 2 hours.

[0069] The present invention provides a monkeypox virus detection kit, comprising a detection test strip and the above-mentioned monkeypox virus detection probe;

[0070] The test paper comprises a sample pad, an NC membrane and an absorption pad. The NC membrane is provided with a T line and a C line. The surface of the T line is coated with monkeypox virus capture antibodies, and the surface of the C line is coated with goat anti-mouse antibodies.

[0071] In the present invention, the method for preparing the test paper preferably comprises the following steps:

[0072] Monkeypox virus capture antibody and goat anti-mouse antibody were added dropwise onto the NC membrane to form T lines and C lines, respectively, to obtain an antibody-coated NC membrane;

[0073] The NC membrane coated with the antibody was dried in a constant temperature oven at 37° C. for 3 h, attached to a PVC backing card, and assembled with a sample pad and an absorption pad to obtain a test paper.

[0074] In the present invention, the monkeypox virus capture antibody is a commercial antibody with the product number 40891-M0027, purchased from Beijing Sino Biological Technology Co., Ltd.; the goat anti-mouse antibody is a commercial antibody purchased from Sangon Biotech (Shanghai) Co., Ltd. with the product number D111024.

[0075] In the present invention, the concentration of the capture antibody added to the T line is preferably 0.5-1.5 mg / mL, more preferably 1 mg / mL; the concentration of the goat anti-mouse IgG added to the C line is preferably 0.5-1.5 mg / mL, more preferably 0.8 mg / mL.

[0076] In the present invention, the obtained test paper is preferably cut to obtain a test paper strip, and the size of the test paper strip is preferably 6.0 cm×0.35 cm.

[0077] The present invention provides a non-diagnostic method for detecting monkeypox virus in a colorimetric-fluorescence dual-signal mode, comprising the following steps:

[0078] Mixing the monkeypox virus detection probe, a buffer solution, and a sample to be tested to obtain a test solution;

[0079] The test solution is added to the sample pad of the test paper, and after standing, the color of the T line on the NC membrane surface is observed. The color depth of the T line is compared with the color of a predetermined colorimetric card to obtain the content of monkeypox virus in the test sample; the colorimetric card is a colorimetric image of the T line on the test paper with different concentrations of monkeypox virus protein.

[0080] The present invention mixes the monkeypox virus detection probe, a buffer solution and a sample to be tested to obtain a test solution. In the present invention, the sample to be tested is preferably a saliva sample or an environmental sample, such as river water.

[0081] In the present invention, the buffer solution is preferably phosphate buffered saline (PBS). In the present invention, the monkeypox virus detection probe is preferably provided in the form of a PBS dispersion; the concentration of the monkeypox virus detection probe PBS dispersion is preferably 1 to 10 mg / mL; and the volume ratio of the monkeypox virus detection probe, buffer solution, and test sample is preferably 1:5 to 10:80 to 100. In the present invention, the mixing method is preferably rotational mixing, and the mixing time is preferably 1 to 20 seconds, more preferably 10 seconds.

[0082] The present invention adds the test solution to the sample pad of the test paper, allows the sample to rest, and then observes the color of the T-line on the surface of the NC membrane. The color depth of the T-line is compared with a predetermined colorimetric chart to determine the monkeypox virus content in the test sample. The colorimetric chart is a colorimetric image of the T-line on the test paper at different concentrations of monkeypox virus protein. In the present invention, the sample volume of the test solution is preferably 80 to 100 μL, and the resting time is preferably 10 to 15 minutes, more preferably 12 to 14 minutes.

[0083] In the present invention, if the sample to be tested contains monkeypox virus, the monkeypox virus detection probe quickly binds to the target antigen to form a virus-labeled immune complex, which is then fixed by the capture antibody pre-coated on the T line, producing a black band visible to the naked eye. If the sample is negative, no monkeypox virus-labeled immune complex can be formed for T-line capture. Therefore, no visible band will appear in the T-line area. The excess immune-signaling label is fixed on the C line using goat anti-mouse antibody. Therefore, a black C line always appears on the NC membrane, indicating the validity of the test.

[0084] In the present invention, the color of the T line on the surface of the NC membrane can also be observed under conditions where the surface of the NC membrane is irradiated with ultraviolet light, and the wavelength of the ultraviolet light is preferably 356 nm. In the present invention, when observed under ultraviolet light excitation conditions, if the sample to be tested contains monkeypox virus, the fluorescence of the T line appears red. The results of the embodiment show that the visual limit of detection (vLOD) of the colorimetric mode for detecting monkeypox virus antigen is 0.1 ng / mL; under ultraviolet light excitation, the visual sensitivity of the fluorescent signal on the T line to the target antigen is 0.01 ng / mL when measured by the naked eye.

[0085] Alternatively, the present invention adds the test solution to the sample pad of the test paper, tests the fluorescence intensity of the T line after standing, and obtains the content of monkeypox virus in the test sample based on the fluorescence intensity and a predetermined standard curve; the standard curve is a linear relationship curve between the monkeypox virus content and the fluorescence intensity.

[0086] In the present invention, the sample volume of the test solution is preferably 80-100 μL; the standing time is preferably 10-15 min, more preferably 12-14 min. The present invention preferably uses a fluorimeter to test the fluorescence intensity, more preferably a portable fluorimeter.

[0087] In the present invention, the method for obtaining the standard curve preferably comprises the following steps:

[0088] Providing a gradient of monkeypox virus antigen solutions with known concentrations;

[0089] The monkeypox virus antigen solution with a known gradient concentration is used as a test sample to prepare a test solution, the test solution is added to the sample pad of the test paper, and the fluorescence intensity of the T line is tested after standing to obtain the fluorescence intensity corresponding to the monkeypox virus antigen solution with different concentrations. The standard curve is drawn with the monkeypox virus antigen solution concentration as the abscissa and the fluorescence intensity as the ordinate.

[0090] As a specific embodiment of the present invention, the standard curve is R 2 =0.998, the linear detection range is 100 ng / mL to 0.01 ng / mL, and the detection limit is 0.0024 ng / mL.

[0091] In the present invention, the preparation process of MoS2-MQDs composite nanomaterials and the principle of colorimetric-fluorescence dual signal mode immunochromatographic detection are as follows Figure 1 shown.

[0092] The following examples describe in detail the molybdenum disulfide-loaded quantum dot shell nanosheets provided by the present invention, their preparation method, and their application in immunochromatography. However, they should not be construed as limiting the scope of protection of the present invention.

[0093] Example 1 Preparation of MoS2-loaded Quantum Dot Shell Nanosheets

[0094] (1) Processing of MoS2 nanosheets:

[0095] Mix 10 mL of a 1 mg / mL MoS2 monolayer solution with flakes of 400-800 nm in diameter with 10 mL of deionized water, sonicate for 10 minutes, and centrifuge. Discard the supernatant containing nanosheets <300 nm in diameter and disperse the MoS2 precipitate in 10 mL of deionized water.

[0096] (2) Preparation of MoS2@PEI nanosheets:

[0097] 10 mL of 2 mg / mL PEI aqueous solution was directly added to the MoS2 solution prepared above, and the reaction was carried out under ultrasonic conditions for 30 min. The excess PEI in the solution was removed by centrifugation.

[0098] (3) Preparation of MoS2-QD composite nanosheets:

[0099] The MoS2@PEI nanosheets were resuspended in 10 mL of deionized water and sonicated for 5 minutes to completely disperse them. The nanosheets were then mixed with 10 nmol of carboxyl-modified CdSe / ZnS QDs under sonication. After 30 minutes, the formed MoS2-QDs were separated by centrifugation and then washed once with deionized water to remove excess QDs.

[0100] (4) Preparation of MoS2-DQDs composite nanosheets:

[0101] The MoS2-QD precipitate was dispersed in 10 mL of deionized water and then reacted with 2 mg / mL 10 mL PEI solution and 10 nmol carboxyl-modified CdSe / ZnS QDs under ultrasound to form MoS2-DQDs with a double-layer QD shell.

[0102] (5) Preparation of MoS2-TQDs and MoS2-FQDs composite nanosheets:

[0103] The layer-by-layer self-assembly steps were repeated to prepare MoS2-TQDs with three QD shells and MoS2-FQDs with four QD shells. The resulting MoS2 composite nanosheets were stored in 10 mL of ethanol for later use.

[0104] Figure 2Transmission electron microscopy (TEM) images and local magnified images of the MoS2 nanosheets obtained by step (1) of this embodiment, the MoS2-QD composite nanosheets obtained by step (3), the MoS2-DQDs composite nanosheets obtained by step (4), and the MoS2-TQDs and MoS2-FQDs composite nanosheets obtained by step (5). Figure a is MoS2, Figure b is MoS2-QD, Figure c is MoS2-DQDs, Figure d is MoS2-TQDs, Figure e is MoS2-FQDs, Figure f, Figure g, Figure h, Figure i and Figure j are magnified TEM images of their local morphologies; Figure k, Figure l and Figure m are typical SEM images of MoS2, MoS2-QD and MoS2-TQD, respectively; Figure n is the EDS element mapping diagram of a single MoS2-TQD nanoparticle and Figure o is the element line scan result.

[0105] The above TEM results show that with the continuous adsorption of 1 to 4 layers of quantum dots, the density of quantum dots in the multilayer shells on the nanosheets increases, indicating that the number of loaded quantum dots increases.

[0106] Figure 3 The fluorescence emission spectrum characterization diagram of the MoS2-MQDs composite nanomaterial of the present invention. It can be seen that similar to the pure MoS2 suspension, the QD-coated MoS2 solution all appears dark gray, indicating its strong colorimetric ability. Under 365nm ultraviolet light excitation, the luminescence ability of free quantum dots, MoS2 and MoS2-QDs with different quantum dot shells was visually compared. The fluorescence signal of MoS2-DQDs, MoS2-TQDs and MoS2-FQDs gradually improved with the increase of the number of QD shell layers on the nanosheet structure. In addition, by comparing the intensity of the maximum emission peak at 631nm, it was found that the luminescence intensity of MoS2-TQDs and MoS2-FQDs was significantly higher than that of MoS2-DQDs, and their enhancement ability was 3.28 times and 4.19 times, respectively. These results show that coating two or more PEI / QD shells on dual-signal NSs can overcome the internal filtering effect of MoS2 sheets and greatly improve the fluorescence ability.

[0107] Figure 4 The fluorescence intensity of the MoS2-MQDs composite nanomaterial at different pH values is shown in Figure 2. It can be seen that the MoS2-MQDs composite nanomaterial exhibits excellent chemical and optical stability in complex environments. These results demonstrate the great potential of dual-signal nanotags for complex sample detection.

[0108] Example 2 Preparation of Monkeypox Virus Detection Probe and Test Paper

[0109] The molybdenum disulfide-loaded quantum dot shell nanosheets (MoS2-QD, MoS2-DQD, MoS2-TQD and MoS2-FQD) prepared in Example 1, anti-monkeypox virus antibodies and a buffer solution were mixed and incubated to obtain a monkeypox virus detection probe.

[0110] Monkeypox virus capture antibody and goat anti-mouse antibody were dripped onto the NC membrane to form T and C lines, respectively. The prepared NC membrane was then dried in a constant temperature oven at 37°C for 3 hours and attached to a PVC backing card, where it was assembled with the sample pad and absorbent pad. Finally, the fully assembled card was cut into individual strips (6.0 cm × 0.35 cm) and stored in a vacuum desiccator until ready for use.

[0111] Example 3

[0112] Four MoS2-based QD tags (MoS2-QD, MoS2-DQD, MoS2-TQD, and MoS2-FQD) were introduced into the same ICA system to determine the effect of multilayer QD shells of dual-signal MoS2-loaded quantum dots on the test strips.

[0113] Add 2 μL of monkeypox virus detection probe and 10 μL of running buffer to 100 μL of the sample to be tested, vortex vigorously for 10 seconds, and then pipette directly onto the ICA test paper sample pad to start analysis. Figure 5 Performance characterization of MoS2 composite nanolabels with different numbers of layers, where Figure a is a photograph (upper figure) and fluorescence image (lower figure) of MoS2-QD-ICA, MoS2-DQD-ICA, MoS2-TQD-ICA and MoS2-FQD-ICA strips used for monkeypox virus detection; Figure b is the corresponding fluorescence intensity on the T line of MoS2-QD-ICA, MoS2-DQD-ICA, MoS2-TQD-ICA and MoS2-FQD-ICA strips; Figure c is the signal-to-noise ratio.

[0114] A dual-signal composite nanotag with multiple QD shells (greater than two) can effectively enhance the fluorescence signal on the ICA strips and improve the fluorescence response sensitivity. Comparisons between different groups showed that the constructed three-layer MoS2-MQDs-ICA exhibited a high fluorescence response on the test strips and achieved the best signal-to-noise ratio for monkeypox virus detection. This MoS2-MQDs-ICA was used in the following examples for related tests.

[0115] Example 4

[0116] In this example, the detection capability (sensitivity and quantitative detection range) of the MoS2-MQDs colorimetric-fluorescence dual-signal ICA was evaluated by detecting monkeypox virus antigen at different concentrations (100-0.005 ng / mL).

[0117] Figure 6 Figure 3 shows the colorimetric images, fluorescence images, and corresponding fluorescence signals on the T line of the monkeypox virus protein test strip at different concentrations. Figure a shows the colorimetric and fluorescence images at different monkeypox virus antigen concentrations (100-0.05 ng / mL), and the detailed fluorescence signal on the T line; Figure b shows the corresponding test strip standard curve.

[0118] As can be seen, the intensity of both the black and red fluorescence signals on the T-line decreases with decreasing concentration of the target viral antigen. Black and red fluorescence are displayed under natural light and 356nm UV excitation, respectively. The results show that the visual limit of detection (vLOD) for monkeypox virus antigen detection using the MoS2-MQDs-ICA colorimetric mode is 0.1 ng / mL. Furthermore, under UV excitation, the visual sensitivity of the fluorescence signal on the T-line to the target antigen is 0.01 ng / mL when measured with the naked eye.

[0119] The fluorescence intensity of the test strips was then analyzed in detail using a handheld dual-mode reader, and the corresponding monkeypox virus calibration curve was drawn ( Figure 6 (b) The fluorescence LOD of monkeypox virus antigen was calculated according to the IUPAC standard protocol to be 0.0024 ng / mL.

[0120] Figure 7 Figure 2 is the characterization of the detection performance of MoS2-MQDs-ICA, where Figure a is the repeatability characterization and Figure b is the specificity characterization. Figure 7 As shown in Figure (a), at the same concentration of viral antigen, all MoS2-MQDs-ICA test strips exhibited a stable and uniform fluorescence signal. The relative standard deviation (RSD) of the fluorescence intensity along the T line was less than 8.75%, demonstrating the high stability and reliability of this method. Figure 7 Figure b shows the specificity of MoS2-TQD for detecting monkeypox virus on ICA strips as shown in this example. It can be seen that cowpox virus, varicella-zoster virus, new coronavirus, influenza A virus, influenza B virus, and adenovirus did not affect the experimental results.

[0121] Figure 8 Figure 2 shows the test results (colorimetric / fluorescence images and corresponding fluorescence signals) for throat swabs and lake water samples spiked with viral antigens. The results show that the colorimetric signals along the T-line of the MoS2-MQDs-ICA strip are essentially consistent with those of the PBS sample, demonstrating that our dual-signal method has good accuracy and applicability for complex real-world samples.

[0122] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A monkeypox virus detection probe comprising a molybdenum disulfide-loaded quantum dot shell nanosheet and an anti-monkeypox virus antibody chemically coupled to the molybdenum disulfide-loaded quantum dot nanosheet; The molybdenum disulfide loaded quantum dot shell nanosheet comprises a MoS2 two-dimensional nanosheet core and several layers of quantum dot shell wrapped on the surface of the core, and the single-layer quantum dot shell comprises a polyethyleneimine layer and carboxylated quantum dots distributed on the surface of the polyethyleneimine layer; The MoS2 two-dimensional nanosheet has a sheet diameter of 400 to 800 nm; The thickness of the single polyethyleneimine layer is 1 to 10 nm; The quantum dot shell has three layers.

2. The monkeypox virus detection probe according to claim 1, wherein The carboxylated quantum dots are one or more of carboxylated CdSe / ZnS quantum dots, carboxylated InP / ZnS quantum dots, and carboxylated carbon quantum dots.

3. The monkeypox virus detection probe according to claim 1, characterized in that The preparation method of the molybdenum disulfide-loaded quantum dot shell nanosheets comprises the following steps: (1) providing a single-layer MoS2 two-dimensional nanosheet dispersion; (2) ultrasonically mixing the single-layer MoS2 two-dimensional nanosheet dispersion with a polyethyleneimine aqueous solution to obtain MoS2@PEI nanosheets; (3) ultrasonically mixing the MoS2@PEI nanosheets and carboxylated quantum dots to obtain MoS2-QD composite nanosheets; (4) Ultrasonic mixing the MoS2-QD composite nanosheets with the polyethyleneimine aqueous solution again, and repeating the process of steps (2) to (3) to obtain molybdenum disulfide-loaded quantum dot shell nanosheets.

4. The monkeypox virus detection probe according to claim 3, characterized in that In the step (2), the concentration of the polyethyleneimine aqueous solution is 0.2 to 5 mg / mL; the mass ratio of the single-layer MoS2 two-dimensional nanosheet to the polyethyleneimine is 1 to 10:1; In the step (3), the molar ratio of the mass of the MoS2 two-dimensional nanosheets to the carboxylated quantum dots is 10 mg:1 to 20 nmol.

5. A monkeypox virus detection kit comprising a test strip and the monkeypox virus detection probe according to claim 1; The test paper comprises a sample pad, an NC membrane and an absorption pad. The NC membrane is provided with a T line and a C line. The surface of the T line is coated with monkeypox virus capture antibodies, and the surface of the C line is coated with goat anti-mouse antibodies.

6. A non-diagnostic method for detecting monkeypox virus using a colorimetric-fluorescence dual-signal mode, comprising the following steps: Mixing the monkeypox virus detection probe according to claim 1, a buffer solution, and a sample to be tested to obtain a test solution; Add the test solution to the sample pad of the test paper, observe the color of the T line on the surface of the NC membrane after standing, compare the color depth of the T line with a predetermined colorimetric card, and obtain the content of monkeypox virus in the test sample; the colorimetric card is a colorimetric image of the T line on the test paper with different concentrations of monkeypox virus protein; Alternatively, the test solution is added to the sample pad of the test paper, and the fluorescence intensity of the T line is tested after standing. The content of monkeypox virus in the test sample is obtained according to the fluorescence intensity and a predetermined standard curve; the standard curve is a linear relationship curve between the monkeypox virus content and the fluorescence intensity.

7. The detection method according to claim 6, characterized in that The color of the T line on the surface of the NC film is observed under the condition that the surface of the NC film is irradiated with ultraviolet light.

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